Abstract

Objective: To establish the prevalence of Fc(epsilon)RI- β polymorphisms Leu181 and Leu181/Leu183 on chromosome 11q13 in the general population and to examine whether when maternally inherited they confer a risk of atopy.

Design: A population based survey for measures of atopy (skin prick test reactions, specific IgE titres, total serum IgE concentration), bronchial hyperresponsiveness, and carriage of Fc(epsilon)RI-β Leu181 and Leu181/Leu183.

Setting: The rural coastal town of Busselton, Western Australia.

Subjects: 1004 members of 230 two generation families identified through adults aged under 55.

Results: Fc(epsilon)RI-β Leu181/Leu183 was identified in 45 subjects (4.5%). All 13 children who had inherited the variant maternally were atopic. Six had asthma and nine rhinitis. The odds ratio of a positive skin prick test reaction to house dust mite or grass pollen in these children compared with the other 523 children was 7.37 (95% confidence interval 1.62 to 33.60). The 95% confidence interval for the odds ratio of a positive specific IgE response (radioallergosorbent test) was 3.00 to (infinity), and the odds ratio for bronchial hyperresponsiveness was 3.70 (1.21 to 11.60). By contrast, the eight children who had derived the variant paternally had negative skin prick and radioallergosorbent test results and did not have increased bronchial responsiveness.

Key messages

Among 1000 members of 230 families from the rural coastal town of Busselton, Western Australia, over 4% were carriers of Leu181/Leu183

When inherited maternally the polymorphism is associated with a significant risk of atopy and bronchial hyperresponsiveness

Leu181/Leu183 is the first genetic risk factor for atopy to be recognised but accounted for only a portion of atopy and asthma in the Busselton population

Introduction

Atopy is a familial syndrome of asthma, rhinitis, and eczema which is due to the interaction of genetic and environmental factors. Genetic linkage of atopic IgE responses to chromosome 11q131 was initially controversial2 but has now been replicated by several groups345 Linkage was strongest in maternally derived alleles,56 which may contribute to the maternal inheritance of atopy evidence from many studies.7891011 The gene for the β chain of the high affinity receptor for IgE (Fc(epsilon)RI-β) is a candidate for the chromosome 11q13 effect.12 Sequencing of Fc-βRI-β has detected polymorphisms (variants)--Fc(epsilon)RI-β Leu181/Leu183 (Leu 181/Leu183) and Fc(epsilon)RI-β Leu181 (Leu181)--which were associated with atopy.13

We examined the prevalence of Leu181 and Leu181/Leu183 in an Australian general population sample and tested whether when inherited maternally the polymorphisms endowed a risk of atopy.

Subjects and methods

The subjects were from the rural coastal town of Busselton, South Western Australia. The aim was to recruit young nuclear families. Children under 5 were excluded because they could not complete respiratory testing. Families were identified through adults aged 55 or under from an electoral roll of around 9000. Families were serially recruited until a predetermined target of 1000 subjects was reached. The final sample comprised 1004 members of 230 nuclear families.

A total of 1867 subjects were approached. Of these, 177 refused to take part; 140 were not contactable; and 434, though contacted, had not been tested by the end of the study. Seven hundred and eight subjects were excluded because their spouses were aged over 55 or because they were not married with two or more natural children over the age of 5. The remaining 408 subjects and their spouses and children not on the electoral roll completed the study. Subjects knew the respiratory interest of the investigation before agreeing to participate. We emphasised that normal people were important to the study.

Clinical protocol--Testing took place in May, June, and July 1992. A respiratory questionnaire based on the American Thoracic Society questionnaire but including questions on rhinitis and allergies was administered. Skin prick testing to house dust mite (Dermatophagoides pteronyssinus), mixed grass pollen, cat and dog dander, Aspergillus fumigatus, Alternaria alternata, and a negative control (Dome-Hollister-Steir, Spokane, United States) was carried out as described.6 Weal diameters were calculated minus the negative control. Bronchial responsiveness to methacholine (maximum dose 12 μmol) was measured.1415 The dose required to provoke a 20% fall in forced expiratory volume in one second was estimated by linear interpolation. Subjects in whom a 20% fall was not achieved by the maximum dose of methacholine were assigned an arbitrary PD of 100.15 Blood was taken by venepuncture for IgE assays, eosinophil and white cell counts, and DNA studies.

DNA testing--DNA was extracted from peripheral blood leucocytes by phenol-chloroform. Fc(epsilon)RI-β Leu181 was detected by the amplification refractory mutation system polymerase chain reaction16 and by sequencing17 (see appendix). Genotyping and phenotyping were performed double blind.

Results

A total of 502 subjects were male. The parents (n=460) were aged 30-55 years (mean 40.2 (SD 4.98) years) and the children (n=544) 5-27 years (mean 12.6 (4.73) years). Forty five per cent of the parents (n=207) and 43% of the children (n=234) had a positive skin prick test reaction >/=4 mm to house dust mite or rye grass or both; 41% of parents (n=189) and 44% of children (n=239) had positive specific IgE titres (radioallergosorbent tests) to either house dust mite or grass pollen or both. Twenty three per cent of the parents (n=106) and 24% of the children (n=131) reported wheezing or whistling in their chest in the previous years, and 8% of the parents (n=37) and 14% of the children (n=76) reported an attack of asthma in the same interval. Half of the parents (n=230) and 42% of the children (n=228) reported episodic sneezing. Forty two per cent of the parents (n=193) and 8% of the children (n=44) had smoked cigarettes.

The assay for Leu181 failed to amplify in five subjects (0.5%). Of the remaining 999 subjects, 45 (4.5%) were positive for Leu181. Twenty one of these were children; eight (seven sibships) had inherited the variant paternally, and 13 (seven sibships) had inherited it maternally. Sequencing of an individual from each family showed that in each case Leu181 was accompanied by Leu183, so that only the Leu181/Leu183 polymorphism was found in this population.

All 13 children who had inherited Leu181/Leu183 maternally were atopic (table I). Eleven had wheeze or rhinitis or both, and a 12th, who denied symptoms, had had asthma previously, diagnosed and treated by a physician. Compared with the 523 children in the population without Leu181/Leu183 the 13 had significantly increased skin prick test reactions and radioallergosorbent test results to house dust mite and grass pollen (table II). The odds ratio for a positive skin prick test reaction >/=4 mm to house dust mite or grass or both, compared with other children, was 7.37 (table III). The 95% confidence interval for the odds ratio of a positive radioallergosorbent test result to either or both allergens was 3.00 to (infinity). When compared only with children with skin prick test reactions >/=4 mm or positive radioallergosorbent test results, or both, children with maternal Leu181/Leu183 still had greater skin prick test reactions and radioallergosorbent test results to house dust mite (P=0.005 and P=0.035 respectively). Hence children with the variant were discernibly more atopic than other children with atopy. The trend was for the total serum IgE concentration to be raised, though the differences were not significant (P=0.08).

TABLE I

Clinical details of children with maternally and paternally inherited Fc(epsilon)RIPβ Leu181/Lei 183

The eosinophil counts in the 13 children were significantly raised, and the PD was significantly lower (table II). Seven children had increased bronchial responsiveness </=10 μmol methacholine14; odds ratio 3.70) (table III).

The eight children who had inherited Leu181/ Leu183 paternally were, by contrast, non-atopic with negative skin prick and radioallergosorbent test results (table I). Skin prick and radioallergosorbent test results and eosinophil counts were less than those of other children (table II). The odds ratio for a positive skin prick test reaction was 0 (95% confidence interval 0 to 0.62; P=0.015), as was the odds ratio for a positive radioallergosorbent test result (0 to 0.36; P=0.002).

Of the 24 parents with Leu181/Leu183, 11 had negative skin prick and radioallergosorbent test results to house dust mite and grass pollen, whereas 13 were positive in both skin prick and radioallergosorbent tests to one or more allergens.

Discussion

Within this sample maternal inheritance of Fc(epsilon)RI-β Leu181/Leu183 was associated with an increased risk of IgE responses to common allergens, raised eosinophil counts, and bronchial hyperresponsiveness. The variant therefore identified a genetic risk factor for atopy.

The study aimed at recruiting nuclear families in whom age had minimally affected measures of atopy and respiratory function. One hundred and seventy seven subjects declined to take part, and participants knew the survey was to study asthma. The sample may therefore not be completely representative, and the estimated 4.5% prevalence of Leu181/Leu183 may be higher than the true value. The high frequency of positive skin prick test results and atopic symptoms is, however, consistent with previous surveys in Busselton18 and other Western populations.1920

The calculations of risk assume independence of observations, though the 13 maternal inheritors of Leu181/Leu183 were from only seven sibships. The odds ratios for the various associations may therefore be somewhat higher than their true value. Nevertheless, the Leu181/Leu183 polymorphism is clearly associated with a clinically significant risk of atopy.

Though Leu181/Leu183 is comparatively rare, it should be considered to be a variant of normal rather than a mutation. The leucine substitutions are conservative but it is not possible to predict whether they are functionally important.2122 It is likely that other common polymorphisms in and around Fc(epsilon)RI-β exist and that haplotypes of these may better define genetic risk due to Fc(epsilon)RI-β.

Leu181/Leu183 was found exclusively in Busselton, though Leu181 seems much more common in native British people.13 This may be due to variation in the prevalence of Fc(epsilon)RI-β polymorphisms between populations. The possibility that the amplification refractory mutation assay failed to amplify Leu181 when it was not accompanied by Leu183 was monitored by the use of positive controls.

In order to interpret the presence of Leu181/Leu183 its parental origin needs to be known. The negative skin test results and specific IgE titres of subjects who had inherited Leu181/Leu183 paternally were unexpected given the high background level of atopy in Busselton. Thirteen of the 24 parents carrying Leu181/ Leu183 were atopic and 11 were not, consistent with active and inactive alleles in the 0.5:0.5 proportion expected from a maternal effect. Possible mechanisms for the maternal effect include genomic imprinting or influences through the placenta or breast milk.6

The timing and degree of exposure to allergen in early life determine the subsequent probability of atopic disease,23 so that recognition of genetic susceptibility may result in prevention of illness.9 The results suggest that polymorphism in Fc(epsilon)RI-β is one factor that can be used to assign genetic risk.

The prevalence of atopy in these subjects cannot be explained alone by the chromosome 11 effect or by variation in Fc(epsilon)RI-β. The TCR-(alpha) region on chromosome 1424 and the interleukin 4 cluster on chromosome 525 also show linkage to IgE responses. Prospective testing for Fc(epsilon)RI-β polymorphisms and examination of the effects of other genes and the environment are required before genotyping at the Fc(epsilon)RI-β locus can be used clinically for genetic risk estimation.

Dr Kevin Cullen pioneered studies of the Busselton population for epidemiological research and contributed to this study until his death on 9 February 1994. We thank the people of Busselton and the volunteers who carried out field testing. Gordon Rich (Western Diagnostic Laboratories) provided laboratory facilities. WOCMC is a Wellcome Trust senior clinical research fellow.

The reaction mixture (40 μl) without enzyme was heated to 95°C for five minutes in a thermal cycler (Hybaid) and held at 80°C for the addition of enzyme (2 units of enzyme in 10 μl reaction buffer). Reaction conditions then followed 35 cycles of 94°C for one minute, 60°C for two minutes, and 72°C for two minutes and one cycle of 72°C for 10 minutes. Amplified products were separated in a 3% (3:1 LMP agarose:Nusieve) gel containing ethidium bromide and visualised under ultraviolet light. Three bands potentially resulted from the primer combinations: 5FU- 3FU gave a 459 bp control band; 5WK-3FU gave a 353 bp band in the presence of the “wild type” Il(epsilon)181; 3M-5FU gave a 163 bp band in the presence of Leu181.

A member of each family segregating Leu181 was sequenced by the method of Sanger et al17 to ensure accuracy of the polymerase chain reaction and to determine whether Leu183 was present. The 459 bp 5FU-3FU band from the above reaction was taken to second round polymerase chain reaction with the following internal primers: 5D (5'biotinylated)--AAG GAC TGG TCA GAT GGT AG;3D--GGC TTC TAT CTA CCT TGT TTC. A single strand template was prepared with strepavidin labelled magnetic beads (Dynal, Oslo, Norway), and direct solid phase sequencing followed with the sequencing primer 3GS-TCC TTT GAG TTC TTC CCC A.

Footnotes

Funding Wellcome Trust.

Conflict of interest The University of Oxford holds a patent protecting the commercial use of Fc(epsilon)RI-β. Two of us (WOCMC and JMH) were coinventors.